The present invention relates to: cells and non-human animals deficient for the protein kinase C isozyme xcex5 (PKCxcex5); the use of PKCxcex5 as a target for drugs; the use of modulators of PKCxcex5 in methods of reducing anxiety, modulating alcohol consumption and self-administration of other drugs of abuse, altering the effects of alcohol, and treating conditions associated with insufficient activity of the GABAA receptor; and the identification of individuals with enhanced susceptibility to alcoholism or other forms of addiction.
Anxiety is very common sensation that, if severe or persistent, can be quite disabling. Anxiety-related disorders are so prevalent that benzodiazepines, the most frequently prescribed anxiolytic agents, regularly appear in lists of the top 20 or 25 most frequently prescribed drugs. Given the undesirable side effects of benzodiazepines and other anxiety-reducing drugs, there is a need for new treatments for anxiety.
Alcoholism is the most common form of drug abuse and a major public health problem worldwide. Nevertheless, few drugs exist that modify alcohol intake and the genetic factors that influence alcohol""s effects on brain and behavioral processes remain largely uncharacterized. Thus, there is a need for diagnostic tests that can identify individuals with a predisposition to becoming alcoholics and a need for treatments that can alter alcohol consumption.
The Lewin Group estimated the economic cost to U.S. society in 1992 due to alcohol and drug abuse to be $246 billion, $148 billion of which was attributed to alcohol abuse and alcoholism and $98 billion of which stemmed from drug abuse and dependence (H. Harwood et al., The Economic Costs of Alcohol and Drug Abuse in the United States, 1992, NIH Publication Number 98-4327 (September 1998)). When adjusted for inflation and population growth, the alcohol estimates for 1992 are very similar to cost estimates produced over the past 20 years, and the drug estimates demonstrate a steady and strong pattern of increase. The current estimates are significantly greater than the most recent detailed estimates developed for 1985 for alcohol and for drugs (Rice et al. 1990)xe2x80x9442 percent higher for alcohol and 50 percent greater for drugs over and above increases due to population growth and inflation
Protein kinase C (PKC) is a multigene family of phospholipid-dependent, serine-threonine kinases central to many signal transduction pathways. So far, ten members, i.e., isozymes, of the PKC family have been described, which are encoded by nine different genes. The ten isozymes are designated as the xcex1-, xcex2I, xcex2II, xcex3-, xcex4-, xcex5-, "xgr"-, xcex7-, "igr"-, and xcex8-isozymes. Nishizuka, 1992, Science 258:607-614; Selbie et al., 1993, J. Bio. Chem. 268:24296-24302. Based on sequence homology and biochemical properties, the PKC gene family has been divided in three groups. A first group, i.e., the xcex1, xcex21, xcex22, and xcex3 isozyme, designated as xe2x80x9cconventionalxe2x80x9d PKCs, are regulated by calcium, diacylglycerol and phorbol esters. A second group, i.e., the xcex4, xcex5, xcex8 and xcex7 isozymes, designated as xe2x80x9cnovel xe2x80x9d PKCs, are calcium-independent, but diacylglycerol and phorbol ester-sensitive. Finally, a third group, i.e., the "xgr", and "igr" isozymes, designated as xe2x80x9catypical xe2x80x9d PKCs, are insensitive to calcium, diacylglycerol, and PMA. In addition, two related phospholipid-dependent kinases, PKCxcexc and protein kinase D, share sequence homology in their regulatory domains to novel PKCs and may constitute a new subgroup. Johannes et al., 1994, J. Biol. Chem. 269:6140-6148; Valverde et al., 1994, Proc. Natl. Acad. Sci. USA 91:8572-8576.
A number of studies with tumor promoting phorbol esters suggest that PKC modulates neural differentiation. For example, phorbol esters induce neural tissue from ectoderm in Xenopus embryos (Otte et al., 1988, Nature 334:618-620) and elicit neurite outgrowth from chick sensory ganglia (Mehta et al., 1993, J. Neurochem. 60:972-98 1, Hsu et al., 1984, Cancer Res. 44:4607-4614), chick ciliary ganglion neurons (Bixby, 1989, Neuron 3:287-297), several human neuroblastoma cell lines (Pahlman et al., 1983, Cell Diff: 12: 165-170; Spinelli et al., 1982, Cancer Res. 42:5067-5073), and rat PC12 cells (Roivainen et al., 1993, Brain Res. 624:85-93; Hall et al. 1988, J. Biol. Chem. 263:4460-4466). Studies using purified isozymes, kinase-defective mutants, and transgenic or mutant cell lines have implicated PKCxcex1, -xcex2, -xcex4, -xcex5, and -"xgr" in the differentiation of nonneural cells (Berra et al., 1993, Cell 74:555-563; Goodnight et al., 1994, Adv. Cancer Res. 64:159-209; Gruber et al., 1992, J. Biol Chem. 267:13356-13360; Macfarlane and Manzel, 1994, J. Biol. Chem. 269:4327-4331; Powell et al., 1992Proc. Natl. Acad. USA 89:146-151). Overexpression of PKCxcex1 or -xcex2 in Xenopus embryos enhances neural induction (Otte and Moon, 1992, Cell 68:1021-1029), but little else is known about the identity of specific PKC isozymes that regulate neural differentiation.
Recent evidence suggests that PKCxcex5 plays a role in neural differentiation and plasticity. PKCxcex5 is expressed predominantly in the nervous system and is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex (Saito et al., 1993, Brain Res. 607:241-248). Within immunoreactive neurons, it is localized to the Golgi apparatus and to axons and presynaptic nerve terminals (Saito et al., supra). PKCxcex5 is activated by growth factors that stimulate neural differentiation such as insulin (Heidereich et al., 1990, J. Biol. Chem. 265:15076-15082) and NGF (Ohmichi et al., 1993, Biochem. J. 295:767-772). In addition, in developing chick brain, it is the major isozyme found in nondividing, differentiating neurons (Mangoura et al., 1993, J. Neurosci. Res. 35:488-498).
Further evidence for involvement of PKCxcex5 in neural differentiation has come from studies with PC12 cells. PC12 cells are derived from neural crest and, when treated with NGF or fibroblast growth factors, undergo dramatic biochemical and morphological differentiation, developing several characteristics of mature sympathetic neurons. Greene et al., 1991, in: Culturing Nerve Cells (Banker, G. and Goslin, K. eds) pp. 207-226, MIT Press, Cambridge, Mass. PKC-activating phorbol esters enhance NGF-induced activation of ERK1 and ERK2 mitogen-activated protein (MAP) kinases and neurite outgrowth in PC12 cells, suggesting that PKC modulates responses to NGF (Rolvainen et al., 1993, supra; Hall et al., 1988, supra; Rolvainen et al., 1995, Proc. Natl. Acad. Sci. USA 92:1891-1895). Studies with ethanol-treated PC12 cells suggested that PKCxcex5 is responsible for this effect. Like phorbol esters, ethanol increases NGF-induced MAP kinase activation and neurite outgrowth through a PKC-dependent mechanism (Roivainen et al., 1993, supra; Roivainen et al., 1995, supra). Ethanol promotes PKC-mediated phosphorylation in PC12 cells by increasing levels of messenger RNA and protein for two PKC isozymes, PKCxcex4 and PKCxcex5 (Messing et al., 1991, J. Biol. Chem., 266:23428-23432; Roivainen et al., 1994, Toward a Molecular Basis of Alcohol Use and Abuse, pp.29-38). Recent data demonstrate that overexpression of PKCxcex5, but not of PKCxcex4, enhances NGF-induced MAP kinase activation and neurite outgrowth (Hundle et al., 1995, J. Biol. Chem. 270:30134-30140). These findings establish PKCxcex5 as a positive modulator of neurite growth. They also suggest that PKCxcex5 mediates the neurite-promoting effect of ethanol and phorbol esters in PC 12 cells.
A recent study suggests that PKCxcex5 specifically mediates enhancement of MAP kinase activation and neurite growth by phorbol esters and ethanol in PC12 cells. PKC activation is generally associated with enzyme translocation to lipid containing structures in particulate fractions of cells. Specifically, studies with PC12 cell lines that stably express the fragments xcex5V1 or xcex4V1, which are derived from the first variable domains of PKCxcex5 or PKCxcex4, showed that each fragment selectively inhibited phorbol ester-induced translocation of its corresponding isozyme, indicating that these fragments can function as isozyme-selective translocation inhibitors. NGF-induced MAP kinase phosphorylation and neurite outgrowth are not enhanced by phorbol esters or ethanol in cells expressing xcex5V1, but they are increased by these agents in cells expressing xcex4V1 and in cells transfected with empty vector.
It has been demonstrated that chronic exposure to ethanol increases total PKC activity, high affinity phorbol ester binding and PKC-mediated phosphorylation in PC12 cells Messing et al., 1991, J. Biol. Chem. 266:23428-23432), which is associated with a selective increase in immunoreactivity and mRNA levels for two PKC isozymes, PKCxcex4 and PKCxcex5 (Roivainen et al., 1994, Protein kinase C and adaptations to ethanol, in: Toward a Molecular Basis of Alcohol Use and Abuse. Jansson B., Jxc3x6rvall H., Rydberg U., Terenius L., and Vallee B. L., eds. Birkhduser Verlag, Basel, 1994. pp. 29-38). Ethanol does not increase diacylglycerol formation in PC12 cells or alter PKC activity in an in vitro assay using a mixture of PKC isozymes partially purified from rat brain. These findings suggest that chronic exposure to ethanol increases PKC activity by increasing expression of PKCxcex4 and PKCxcex5. Further, it has been demonstrated that PKCxcex5 is involved in two ethanol-induced processes in PC12 cells; first, it has been shown that ethanol potentiates NGF-induced activation of mitogen-activated protein kinases and neurite outgrowth in PC12 cells by a PKCxcex5-dependent mechanism (Roivainen et al., 1993, Brain Res. 624:85-93, Roivainen et al., 1995, Proc. Natl. Acad. Sci. USA 92:1891-1895; Messing et al., 1991, Brain Res. 565:301-311); second, evidence suggesting that ethanol increases the number of N-type voltage gated Ca2+ channels in PC12 cells and rodent brain by a PKCxcex5-dependent process (Messing et al., Alcoholism Clinical and Experimental Research 22: Abstract S26:2 (1998)). Since both neural plasticity (Robinson and Kolb, 1998, J. Neurosci. 17:8491-8497) and increases in the activity of Ca2+ channels (Messing and Diamond, 1997, Molecular biology of alcohol dependence, in: The Molecular and Genetic Basis of Neurological Disease. Rosenberg R., Prusiner S., DiMauro S., and Barchi R., eds. Butterworth-Heinemann, Boston, pp. 1109-1126) may contribute to drug dependence, PKCxcex5 may have a behavior-modulating effect.
GABA (gamma amino butyric acid) is the major inhibitory neurotransmitter in the brain and GABAA receptors are receptor-gated chloride channels. Upon binding GABA, these channels open, allowing chloride to pass in or out of the cell. This tends to hold the membrane potential of the cell at negative values close to the resting membrane potential, thereby preventing the generation of an action potential. Benzodiazepines are a class of drugs commonly used to reduce anxiety. Benzodiazepines bind with high affinity to GABAA receptors in the central nervous system. DeLorey and Olsen, 1992, J. Biol. Chem. 267:16747-16750. Pentobarbital and benzodiazepines such as diazepam allosterically regulate the GABAA receptor channel, increasing the Cl31  channel open time or the probably of channel opening in response to GABA (A. Guidotti, M. G. Corda, B. C. Wise, F. Vaccarino, E. Costa, Neuropharmacology 22, 1471-9 (1983)). GABA-dependent neurotransmission is thereby enhanced. In contrast, muscimol binds competitively to the GABA recognition site on GABAA receptors and can elevate Clxe2x88x92 conductance independently of endogenous GABA.
Previous studies have provided conflicting reports regarding PKC regulation of GABAA receptors. GABAA receptors are heteropentameric complexes of related subunits, several of which contain consensus sequences for PKC phosphorylation. Moss, 1992, J. Biol. Chem. 267:14470-14476. The xcex32 subunit of the GABAA receptor exists in two forms produced by alternate splicing of mRNA, and some studies suggest that the long splice variant (xcex32L), which contains a unique consensus site for PKC phosphorylation, is specifically required for ethanol sensitivity of GABAA receptors (Wafford et al., 1990, Science 249:291-293; K. A. Wafford, et al., Neuron 7, 27-33 (1991); K. A. Wafford, P. J. Whiting, Febs Letters 313, 113-7 (1992)). However, others have failed to observe this requirement (W. Marszalec, Y. Kurata, B. J. Hamilton, D. B. Carter, T. Narahashi, Journal of Pharmacology and Experimental Therapeutics 269, 157-63 (1994); E. Sigel, R. Baur, P. Malherbe, FEBS Letters 324, 140-142 (1993); D. W. Sapp, H. H. Yeh, Journal of Pharmacology and Experimental Therapeutics 284, 768-76 (1998)), and mice lacking xcex32L show normal behavioral and electrophysiological responses to ethanol (G. E. Homanics, J. J. Quinlan, R. M. Mihalek, L. L. Firestone, Frontiers in Bioscience 3, D548-58 (1998)). Phorbol ester treatment of mouse cerebellar microsacs or of Xenopus oocytes and human kidney cells expressing GABAA receptor subunits inhibits receptor activation by GABA or muscimol (B. J. Krishek, et al., Neuron 12, 1081-95 (1994); N. J. Leidenheimer, R. A. Harris, Advances in Biochemical Psychopharmacology 47, 269-79 (1992); S. Kellenberger, P. Matherbe, E. Sigel, The Journal of Biological Chemistry 267, 25660-25663 (1992)). In contrast, an active catalytic domain of PKC enhances GABA-stimulated currents when expressed in fibroblasts or microinjected into CA1 hippocampal pyramidal neurons (P. Poisbeau, M. C. Cheney, M. D. Browning, I. Mody, Journal of Neuroscience 19, 674-83 (1999); Y. F. Lin, M. D. Browning, E. M. Dudek, R. L. Macdonald, Neuron 13, 1421-1431 (1994)).
As discussed above, prior to the present invention, little was known about the role of PKCxcex5 in vivo in alcoholism, anxiety, drug abuse or GABAA receptor function.
To study the role of PKCxcex5 in vivo in alcoholism, anxiety, drug abuse, GABAA receptor function, and other processes, the inventors have used gene targeting by homologous recombination to create mutant mice that lack PKCxcex5.
The present invention relates, inter alia, to: 1) the production of PKCxcex5 deficient cells and non-human animals; 2) the identification and the use of the PKC isozyme xcex5 (PKCxcex5) as a target for the modulation of anxiety in a mammal; 3) the use of modulators of PKCxcex5 to modulate alcohol consumption and self-administration of other drugs of abuse and the effects of alcohol and other drug consumption; 4) the use of inhibitors of PKCxcex5, either alone or in conjunction with allosteric agonists of GABAA receptors, to treat conditions, such as anxiety, addiction, withdrawal syndrome, skeletal muscle spasms, convulsive seizures, and epilepsy, that are amenable to treatment by allosteric agonists of GABAA receptors; and 5) a diagnostic method for identifying individuals at risk for becoming alcoholics or abusers of other drugs.
The present invention is based, in part, on the inventors"" discovery that PKCxcex5xe2x88x92/xe2x88x92 mice have less fear and anxiety than wild-type mice. This suggests that PKCxcex5 is a target for the development of anxiety-reducing drugs. Furthermore, the invention is based, in part, on the inventors"" discovery that PKCxcex5xe2x88x92/xe2x88x92 mice sleep twice as long as wild-type mice when injected intraperitoneally with drugs that act at GABAA receptors, such as ethanol, pentobarbital or benzodiazepines. This result suggests that PKCxcex5xe2x88x92/xe2x88x92 mice are hypersensitive to the sedative-hypnotic effects of compounds acting at GABAA receptors. Thus, inhibition of PKCxcex5 augments GABAA receptor-mediated signaling, and based on the fact that GABAA agonists are anxiolytics, it can be concluded that PKCxcex5 inhibitors are potent suppressors of anxiety. This conclusion is supported by the observation that PKCxcex5xe2x88x92/xe2x88x92 mice have reduced basal levels of stress-associated hormones and accelerated reduction of hormone levels in the wake of an event that increases such levels.
In one specific aspect, the present invention is directed to animal cells that are PKCxcex5 deficient due to a disruption in the PKCxcex5 coding nucleic acid sequences. An additional aspect of the present invention is the use of a genetically modified PKCxcex5 deficient cell to generate PKCxcex5 -deficient non-human transgenic embryos and animals. Other aspects of the present invention are the PKCxcex5-deficient non-human, preferably mouse, transgenic embryos and animals, and offspring that comprise a targeted disruption in the PKCxcex5 gene, and hence produce less than wild-type levels of PKCxcex5 activity. The PKCxcex5 deficient non-human transgenic animals of the present invention may be heterozygous or homozygous for the mutated PKCxcex5 allele.
The present invention is also directed to assays for identifying anxiolytic compounds. The assays of the invention comprise identification of a compound that inhibits the enzymatic activity of PKCxcex5, and isolation of such compound. In another specific aspect, the present invention is directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound inhibiting the enzymatic activity of PKCxcex5 and a pharmaceutically acceptable carrier. In addition, the present invention is directed to the treatment of anxiety by administration of such pharmaceutical compositions.
Other aspects of the present invention are methods of modulating consumption of a drug of abuse and/or the effects of such drug by administering a modulator of PKCxcex5. Administration of an inhibitor of PKCxcex5 would thereby reduce consumption of alcohol, barbituates, nicotine, opiates, or psychostimulants. Increased consumption of such drugs would result from embodiments of the method that involve the administration of enhancers of PKCxcex5.
Another aspect of the present invention is based on the discovery that PKCxcex5 acts as a selective modulator of endogenous and nonendogenous allosteric agonists of GABAA receptors. Therefore, conditions that are amenable to treatment by such allosteric agonists of GABAA receptors can be treated by methods of the present invention that involve the administration of an inhibitor of the xcex5 isozyme of protein kinase C (PKCxcex5) alone or in combination with such allosteric agonists. Conditions suitable for such treatment include anxiety, addiction, withdrawal syndrome, skeletal muscle spasms, convulsive seizures, and epilepsy. A composition comprising an inhibitor of PKCxcex5 and an allosteric agonist of a GABAA receptor is yet another aspect of the present invention. An additional aspect of the present invention is a method for reducing the effective dose of an allosteric agonist of a GABAA receptor by administering an inhibitor of the xcex5 isozyme of protein kinase C (PKCxcex5) in combination with such allosteric agonist.
Further aspects of the present invention include diagnostic methods and kits for predicting the likelihood that a person will become addicted to a drug of abuse.